The present invention is directed toward immobilized lipid-bilayer materials and, more particularly, to encapsulated lipid-bilayer materials prepared using a sol-gel method. The encapsulated lipid-bilayer materials are useful in fluorimetric methods for detecting metal ions, as a drug delivery system and in separation science.
Lipid-bilayer materials can perform as sensitive optical sensors for the detection of target analytes, such as heavy metal ions. Lipid-bilayer materials exhibit remarkable response times, selectivity, and easily monitored optical signals. They are also simple in design, inexpensive and easy to fabricate. As free-floating aggregates in solution, detectors utilizing lipid-bilayer materials can be used as simple assay systems (U.S. Pat. No. 5,616,790 issued to Arnold et al. on Apr. 1, 1997). An improvement, however, would be to configure these materials to various solid sensor platforms, offering the advantages of further chemical and physical stabilization of the lipid-bilayer materials and allowing facile handling and the opportunity to recover and reuse them. Still, this effort has been frustrated by the difficulty in immobilizing lipid assemblies to surfaces.
Lipid bilayers are aggregates of lipids held together by hydrophobic interactions that form a variety of different structures, such as closed spherical liposomes (or vesicles), flat discs, globules, tubes, and helices (Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709). Due to their dynamic, self-assembled nature, lipid bilayer assemblies are inherently difficult to fix to solid surfaces. The weak forces that create lipid bilayers are easily disrupted by surface modifications and the lipophilic/hydrophilic balance in the surrounding environment. Surface fixation of liposomes and other lipid-bilayer materials typically results in deformation or lysis of the membrane.
Methods have been developed that overcome some of the difficulties in immobilizing the lipid to a surface without damaging the lipid by using biologically based polysaccharides and biocompatible acrylate gels. For example, entrapped lipid bilayer materials, specifically liposomes, in polysaccharide gels have been used for anesthesia sensors, drug delivery systems, biological studies of membrane proteins and phospholipids, chromatography, and biosensors. However, low liposome entrapment volume, the inability to immobilize pre-formed liposomes, and material instability at elevated temperatures are some of the shortcomings that have not been resolved. Liposome entrapment procedures for these polysaccharide and acrylate gels use both an "in situ entrapment" (Merlo, S.; Yager, P. Anal. Chem. 1990, 62, 2728) which forms the gel around the liposomes and a "pore entrapment" of liposomes in pre-formed gels (Lundahl, P.; Yang, Q.; Greijer, E.; Sandbert, M., Immobilization of Liposomes in Gel Beads; 2.sup.nd ed.; CRC Press, 1993; Vol. 1, 343-61). The "in situ" procedure calls for elevated temperatures to dissolve the polysaccharides prior to gelation. The liposomes must endure the elevated temperatures during the encapsulation, which means that only thermally stable liposomes can be entrapped. The "pore entrapment" procedure fills large pores in sepharose gels beads with liposomes to be subsequently sealed in the pore. This technique leads to inherent problems with liposome fusion events and poor homogeneous dispersion of liposomes in the matrix, which can lead to problems with sensor and drug delivery applications.
Another difficulty that exists with these organic based immobilization matrices is the potential for biological digestion of the matrix and lipid bilayers by bacteria, fungi, and molds. The organic matrices of collagen gels (Weiner, et al., J. of Pharmaceutical Sciences, 1985, 74, 922), gelatins (Vestar, Europe Patent 0162724), polyacrylates, and DNA (Fabre, P. et. al. U.S. Pat. No. 5,376,379.) materials are ideal hosts for biological growth. In a controlled laboratory environment the gels can be kept in sterilized conditions. However, use of these materials in the natural environment will lead to rapid degradation of the matrix and bilayer assemblies from the infection and colony growth of the biological "predators".
A non-organic gel-type technique has been developed using phosphazene polymers for liposome encapsulation (Cohen, S.; Bano, M. C.; Visscher, K. B.; Chow, M.; Allcock, H. R.; Langer, R., J. Am. Chem. Soc., 1990, 112, 7832). In this procedure, the phosphazene polymer is functionalized with carboxylic acid residues and dispersed in water. Adjustment of the solution pH to 7.5-7.8 generates an ionically cross-linked matrix that can be used to encapsulate bio-materials. A significant drawback to this technique is the narrow pH range of operation beyond which the encapsulation is lost.
Other existing technology can be found in several areas that relate to self-assembled systems in solid materials. Polymerized lipid tubules, formed by aggregation of diacetylenic lipids followed by photopolymerization, have been easily prepared as composite materials with epoxy resins (Schnur, J. M, Science, 1993, 262, 1669). These lipid tubules are, however, polymerized through the diacetylenic functionality that stabilizes the lipid aggregates with strong covalent interactions. Such lipid assemblies are very solvent and temperature durable as is evident in the composite processing which involves acetone suspension of tubules and high temperature/magnetic field material fabrication.
Whole cells have been entrapped in sol-gel materials via similar techniques as described herein, with cellular function maintained (U.S. Pat. No. 5,200,334 issued to Dunn et al. on Apr. 6, 1993; U.S. Pat. No. 5,300,564 issued to Avnir et al. on Apr. 5, 1994; Carturan, C.; Campstrini, R.; Dire, S.; Scardi, V.; DeAlteriis, E., J. Molec. Catalysis, 1989, 57, L13). However, although whole cells consist of membranes similar to liposomes, living cells are inherently robust structures stabilized by membrane imbedded proteins that organize membrane lipids and are supported by a bio-polymer mesh (actin fibers) that gives the cellular membrane structure and stability. Similarly, encapsulation of enzymes and other proteins (Ellerby, et al., Reports, 1992, 1113-1115) is significantly easier to accomplish because of the more robust structures of the proteins. Simple, lipid-only aggregates containing no supporting framework, such as lipid-bilayer materials, are significantly more fragile to immobilization techniques compared to whole cells.
Drug delivery systems based on liposomes have shown promise as drug carriers in animal and human studies (U.S. Pat. No. 4,740,375 issued to Geho et al. issued on Apr. 26, 1988; U.S. Pat. No. 4,921,257 issued to Wheatley et al. issued on May 1, 1990). However, the liposomes are rapidly removed from the body's blood stream by the spleen and liver as the body recognizes them as foreign invaders. Progress has been made in the circulation time of liposomes in the blood by disguising them with polyethyleneoxide ligands that serve as a "stealth" coating (Lasic, D. D., Angew. Chem., Int. Ed. Engl., 1994, 33, 1685).
Lipid bilayer mimics have recently been used as unique separation materials for peptides, proteins, nucleotides, and oligonucleotides. Lecithin-like molecules were covalently attached to silica particles in an effort to prepare membrane mimics for columns. This support is termed immobilized artificial membranes (IAMs) (Pidgeon, C.; Venkataram, U. V.; Anal. Biochem., 1989, 176, 36). These lecithin-covered column supports could separate biological molecules under milder conditions and with better resolution than conventional chromatographic supports. Furthermore, these supports were very mild to proteins (e.g., cytochrome p450) during separation resulting in no loss of biological activity. Conventional supports often denature the proteins yielding solutions with less than 1 % activity as that prepared from the IAM columns. Materials such as lipid bilayers could also be used as separation materials provided they could be suitably immobilized and fixed to a support.